Quantile-specific heritability of plasma fibrinogen concentrations

Background Fibrinogen is a moderately heritable blood protein showing different genetic effects by sex, race, smoking status, pollution exposure, and disease status. These interactions may be explained in part by “quantile-dependent expressivity”, where the effect size of a genetic variant depends upon whether the phenotype (e.g. plasma fibrinogen concentration) is high or low relative to its distribution. Purpose Determine whether fibrinogen heritability (h2) is quantile-specific, and whether quantile-specific h2 could account for fibrinogen gene-environment interactions. Methods Plasma fibrinogen concentrations from 5689 offspring-parent pairs and 1932 sibships from the Framingham Heart Study were analyzed. Quantile-specific heritability from offspring-parent (βOP, h2 = 2βOP/(1+rspouse)) and full-sib regression slopes (βFS, h2 = {(1+8rspouseβFS)0.05–1}/(2rspouse)) were robustly estimated by quantile regression with nonparametric significance assigned from 1000 bootstrap samples. Results Quantile-specific h2 (±SE) increased with increasing percentiles of the offspring’s age- and sex-adjusted fibrinogen distribution when estimated from βOP (Ptrend = 5.5x10-6): 0.30±0.05 at the 10th, 0.37±0.04 at the 25th, 0.48±0.05 at the 50th, 0.61±0.06 at the 75th, and 0.65±0.08 at the 90th percentile, and when estimated from βFS (Ptrend = 0.008): 0.28±0.04 at the 10th, 0.31±0.04 at the 25th, 0.36±0.03 at the 50th, 0.41±0.05 at the 75th, and 0.50±0.06 at the 90th percentile. The larger genetic effect at higher average fibrinogen concentrations may contribute to fibrinogen’s greater heritability in women than men and in Blacks than Whites, and greater increase from smoking and air pollution for the FGB -455G>A A-allele. It may also explain greater fibrinogen differences between: 1) FGB -455G>A genotypes during acute phase reactions than usual conditions, 2) GTSM1 and IL-6 -572C>G genotypes in smokers than nonsmokers, 3) FGB -148C>T genotypes in untreated than treated diabetics, and LPL PvuII genotypes in macroalbuminuric than normoalbuminuric patients. Conclusion Fibrinogen heritability is quantile specific, which may explain or contribute to its gene-environment interactions. The analyses do not disprove the traditional gene-environment interpretations of these examples, rather quantile-dependent expressivity provides an alternative explanation that warrants consideration.

Traditional genetic analyses assume that the effect of a gene is a constant throughout its distribution. Alternatively, quantile-dependent expressivity occurs when the phenotypic expression of a gene depends upon the level of the phenotype, i.e., whether the trait (e.g., plasma fibrinogen concentration) is relatively high or low [33][34][35][36][37][38][39][40][41]. We have shown that the heritability of adiposity [33,34], lipoprotein cholesterol [33,35], triglyceride [33,38], and adiponectin concentrations [37], and usual coffee [40] and alcohol intake [41] are all quantile-dependent. Specifically, heritability estimated from their offspring-parent and full-sib regression slopes increased significantly from the lowest to the highest percentiles of each trait's distribution. Moreover, within individuals, the genetic effects size of many triglyceride-related single nucleotide polymorphisms (SNP) change over time during postprandial lipemia, i.e., in relation to Information Coordinating Center at the above website, where they can find information on human use approval and data use agreement requiring signature by an official with signing authority for their institute. The authors did not have special access to the data that other researchers would not be allowed to obtain. The public summary-level phenotype data may be browsed at the dbGaP study home page [34].
the changing average triglyceride concentrations following fat ingestion [39]. The hypothesis may be supported in part by Porcu et al.'s finding that differentially expressed genes may reflect disease-induced rather than disease-causing changes in the transcriptome [42].
If the heritability of plasma fibrinogen concentrations is quantile-specific, then this could contribute to some of the gene-environment interactions reported for fibrinogen. Specifically, subjects selected for characteristics that distinguish high vs. low concentrations (e.g., smoking vs. nonsmoking) would be expected to have different size genetic effects. We therefore sought to test whether estimated heritability of fibrinogen concentrations from offspring-parent pairs and full sibs were quantile-specific in a large population cohort (Framingham Heart Study [43,44]). Heritability was analyzed because 47 independently significant variants at 41 independent loci collectively explained only about 3% of the variance of fibrinogen concentrations [45], i.e. a very small percentage of fibrinogen's heritability. We also re-assessed published reports of fibrinogen gene-environment interactions from the perspective of quantile-dependent expressivity. The results suggest that quantile-dependent expressivity: 1) may contribute to the greater fibrinogen heritability in women than men [46]

Methods
The data were obtained from the National Institutes of Health FRAMCOHORT, GEN3, FRA-MOFFSPRING Research Materials obtained from the National Heart Lung and Blood Institute (NHLBI) Biologic Specimen and Data Repository Information Coordinating Center. The hypothesis tested are exploratory and not considered as part of the initial Framingham Study design.
Subjects were at least 16 years of age and not self-identified as nonwhite or Hispanic. Fibrinogen concentrations were measured for examinations 5,6, and 7 of the Offspring Cohort, and examination 1 of the Third Generation Cohort. Venous blood samples were drawn in the morning into a 3.8% sodium citrate solution with a blood/sodium citrate ratio of 9:1 in volume, which was then centrifugation at 2000xg to produce platelet-poor plasma that was stored at -80˚C. Plasma fibrinogen concentrations were measured by the Clauss method (Diagnostica Stago Reagents) [60], which showed intra-assay and inter-assay coefficients of variation of 2.6% and 4.7%, respectively.
Our analyses of these data were approved by Lawrence Berkeley National Laboratory Human Subjects Committee (HSC) for protocol "Gene-environment interaction vs. quantiledependent penetrance of established SNPs (107H021)". LBNL holds the Office of Human Research Protections Federal wide Assurance number FWA 00006253. The protocol has approval number: 107H021-13MR20. All surveys were conducted under the direction of the Framingham Heart Study human use committee guidelines, with signed informed consent from all participants or parent and/or legal guardian if <18 years of age.
Simultaneous quantile regression was performed using the "sqreg" command of Stata (version. 11, StataCorp, College Station, TX) with one thousand bootstrap samples drawn to estimate the variance-covariance matrix for the 91 quantile regression coefficients (β FS ) between the 5 th and 95 th percentiles, and the post-estimation procedures (test and lincom) to test linear combinations of the β FS slopes after estimation with S(k i -1) degrees of freedom. Quantile-specific offspring-parent concordance was assessed by: 1) estimating quantile-specific β OP -coefficient for the 5 th , 6 th , . . ., 95 th percentiles of the sample distribution using simultaneous quantile regression (the <5 th and >95 th percentiles ignored because they were thought to be less stable); 2) plotting the quantile-specific β OP coefficients vs. the percentile of the trait distribution; and 3) testing whether the resulting graph is constant, or changes as a linear, quadratic, or cubic function of the percentile of the trait distribution using orthogonal polynomials [64].
Heritability in the narrow sense (h 2 ) was estimated as h 2 = 2β OP /(1+r spouse ) from offspringparent regression slopes (β OP ) and h 2 = {(1+8r spouse β FS ) 0.5 -1}/2r spouse from full-sibs regression slopes (β FS ) where r spouse is the spouse correlation [65]. "Quantile-specific heritability" refers to the heritability statistic (h 2 ), whereas "quantile-dependent expressivity" is the biological phenomenon of the trait expression being quantile-dependent. In some cases, published reports of geneenvironment interactions were originally presented as histograms or line graphs [51,52,55,66], which were imported into Microsoft PowerPoint (version 12.3.6 for Macintosh computers, Microsoft corporation, Redmond WA) to extract their quantitative information as previously described [39]. Means, slopes, and h 2 are presented with their standard errors (±SE).
Data availability: The data are not being published in accordance with the data use agreement between the NIH National Heart Lung, and Blood Institute and Lawrence Berkeley National Laboratory. However, the data that support the findings of this study are available from NIH National Heart Lung, and Blood Institute Biologic Specimen and Data Repository Information Coordinating Center directly through the website https://biolincc.nhlbi.nih.gov/ my/submitted/request/ [67]. Restrictions apply to the availability of these data, which were used under license for this study. Those wishing a copy of the data set should contact the Blood Institute Biologic Specimen and Data Repository Information Coordinating Center at the above website, where they can find information on human use approval and data use agreement requiring signature by an official with signing authority for their institute. The public summary-level phenotype data may be browsed at the dbGaP study home page [68].

Results
Offspring-parent regression analyses were performed on 1551 offspring with one parent and 2069 with both parents. Full-sib regression analysis were performed using 2045 siblings in 778 sibships from the Offspring Cohort, and 3334 siblings from 1154 sibships from the Third Generation Cohort. The characteristics for the participants used in the analyses are presented in Table 1. As expected, average plasma fibrinogen concentrations were higher for females than males in both cohorts.

Quantile-dependent expressivity
Fig 1A presents the offspring-parent regression slopes (β OP ) at the 10 th , 25 th , 50 th , 75 th , and 90 th percentiles of the offspring's age-and sex-adjusted fibrinogen distribution. The slopes, and their corresponding heritability estimates (h 2 = 2 � β OP /(1+r spouse )), get progressively steeper with increasing percentiles of the distribution. The heritability at the 90 th percentile was 2.2-fold greater than the heritability at the 10 th percentile (P difference = 5.6x10 -5 ). Specifically, the figure shows that h 2 increased from 0.295±0.047 at their 10th percentile (P = 4.5x10 -10 ), 0.374±0.040 at the 25th (P<10 −15 ), 0.479±0.045 at the 50th (P<10 −15 ), 0.605±0.056 at the 75th (P<10 −15 ), and 0.648±0.082 at the 90th percentile of the offspring' distribution (P<3.3x10 -15 ). These slopes, along with those of the other percentiles between the 5 th and 95 th percentiles are presented in the quantile-specific heritability plot in Fig 1B. The corresponding h 2 are displayed on the right. They show heritability increased linearly with increasing percentiles of the offspring's distribution (i.e., slope±SE: 0.0040±0.0009 for each percent increment, P linear = 5.5x10 -6 ) with no significant evidence of nonlinearity (i.e., P quadratic = 0.41; P cubic = 0.82). Quantile-specific heritability was significant (P�1.8x10 -6 ) for all 91 individual percentiles between the 5 th and 95 th percentiles of the offspring's distribution. If the classical model of constant heritability over all quantiles applied, then the line segments in Fig 1A would be parallel, and the graph in Fig 1B would be flat (i.e., zero slope). Heritability estimated from the offspring-midparental slope also increased linearly with increasing percentiles of the fibrinogen distribution (0.0033±0.0009, P linear = 0.0005, not displayed). Fig 2 displays the quantile regression analysis for h 2 estimated from full-sib regression slopes (β FS ). Each one-percent increase in the fibrinogen distribution was associated with a 0.0026±0.0010 increase in heritability and a 0.0013±0.0005 increase in the full-sib regression slope (P linear = 0.008).

Gene-environment interaction
Quantile-dependent expressivity may contribute to gene-environment interactions reported for plasma fibrinogen concentrations. Specifically, the selection of subjects by characteristics that distinguish high from low fibrinogen concentrations (e.g., smokers vs. nonsmokers, exposed vs. unexposed to pollution, untreated vs. treated T2DM) is expected to create different  genetic effects that might be simply explained by quantile-specific heritability (Fig 1). The examples that follow present genotype-specific effects of environmental factors on fibrinogen concentrations (a precision medicine interpretation) that quantile-dependent expressivity would attribute to a larger genetic effect size at higher average fibrinogen concentrations. The analyses do not disprove the traditional gene-environment explanation these interactions, rather quantile-dependent expressivity provides an alternative explanation that warrants consideration.

Sex and ethnicity
For example, meta-analyses suggest that fibrinogen concentrations are 0.16 g/L higher in women than in men and 0.12 g/L higher in Blacks than Whites [1,5] P<0.0001). Some of these sex and racial difference could be the consequence of quantiledependent expressivity, i.e., larger genetic effect size at higher concentrations.

Smoking
Smoke-induced inflammations of the lungs and other organs increase proinflammatory cytokines and circulating inflammatory markers [5]. , consistent with the dose-related trend between fibrinogen concentrations and daily cigarette use [5].
Glutathione-S-transferases (GTS) are phase II enzymes that catalyze glutathione's reaction with dichloromethane and polycyclic aromatic hydrocarbons in cigarette smoke to produce inactive and non-toxic hydrophilic conjugates. Miller et al. [51] reported that the affect of smoking more vs. less than 20 pack-years on plasma fibrinogen concentrations was greater for the M1-0 (null) than M1-1 (functional) genotypes of the GTSM1 polymorphism (Fig 3C, P interaction = 0.06). However, average fibrinogen concentrations were significantly greater in the � 20 pack-year smokers (3.18 vs. 2.97 g/L) and quantile-dependent expressivity would attribute some of the interaction to the larger genetic effect size (0.335 vs. 0.106 mg/dL) at the higher average fibrinogen concentrations of the more-exposed smokers, i.e., a greater impact of not detoxifying toxins at higher fibrinogen concentrations [51].
Interleukin-6 (IL-6) is an important upstream messenger cytokine in inflammation that regulates hepatic fibrinogen production by stimulating FGA, FGB, and FGG promoter regions. It is the primary mediator of acute phase-induced fibrinogen synthesis [70]. The IL-6 -572C>G polymorphism is a relatively common allele in Koreans affecting transcription strength. Results presented by Shin et al.
[52] (their Fig 2) suggest a somewhat greater effect of smoking on the average fibrinogen concentrations in GG homozygotes than carriers of the C allele (Fig 3D histogram) which quantile-dependent expressivity would attribute to a greater effect of the -572C>G polymorphism at higher fibrinogen concentrations (Fig 3D line graph).

Pollution
Fibrinogen concentrations are higher after exposure to traffic pollutants [8]. This may be due to particles deposited in the lungs inducing alveolar inflammation and the cytokine release causing systemic inflammation. Bind et al. [53] reported that the percent change in fibrinogen concentrations associated with air pollution particle number was significantly greater for a high vs. low genetic risk score (P = 0.04). Peters et al. [54] reported that the increase in fibrinogen concentrations with increasing PM 10 concentrations (i.e., particulate matter with an aerodynamic diameter <10 μm) was 8-fold greater in -455 AA homozygotes than GG homozygotes (P interaction = 0.04). The authors interpreted their results from the perspective of genotype-specific susceptibility to ambient particulate matter, where the risk alleles produced an augmented response to environmental inflammatory stimuli in addition to constitutionally higher fibrinogen concentrations. The risk alleles were hypothesized to affect early physiological responses such fibrinogen transcription. Alternatively, an interpretation from the perspective of quantile-dependent expressivity would argue that environmental inflammatory stimuli increase average fibrinogen concentration, which in turn, accentuated the genetic effects. Instead of being a susceptibility locus, FGB -455G>A may simply track the increasing heritability at higher fibrinogen concentrations.

Acute-phase response
Increases the hepatic synthesis and release of fibrinogen and other acute-phase reactants may be triggered by specific inflammatory cytokine signals following severe injury or exercise recovery [71]. C-reactive protein (CRP) is both an acute-phase protein and a general marker for inflammation. Gardemann et al. reported that the difference between FGB -455 AA and GG homozygotes was almost three-fold greater in subjects with clinical signs of an acute phase reaction (i.e., a CRP� the 50 th percentile) than without, consistent with the higher mean fibrinogen concentrations during the reaction (Fig 3E) [55]. They also reported that AAhomozygotes showed significantly greater fibrinogen increases 2-days after aortacoronary bypass surgery than carriers of the G-allele (Fig 3F histogram), which the line graph suggests could be simply the consequence of the higher fibrinogen concentrations two days post-surgery. Another study by Ferrer-Antunes et al. [56] concluded that the increase of fibrinogen level in acute phase situations like severe trauma is associated with the β-gene -455G/A polymorphism (Fig 4A histogram). Again, the line graph shows that following acute cranial or thoracic trauma, the differences between -455 A-allele carriers vs. GG homozygotes were greater at peak concentrations (0.77±0.34 g/L) when average fibrinogen concentrations were highest (5.47±0.17 g/L) than when first measured (effect size 0.14±0.40 g/L) when average concentrations were lower (3.86±0.20 g/L).

Coronary heart disease
In the ECTIM Study, Behague et al. [49] reported that the affect of the FGB -455G/A polymorphism on plasma fibrinogen concentrations appeared to be stronger in cases having a prior history of definite acute MI than those without a history, particularly in smokers [50], consistent with the cases' higher average fibrinogen concentrations (3.44±0.04 vs. 3.00±0.03 g/L, Fig 4C).

Diabetes
Ceriello et al. [58] reported that intensive insulin therapy in T2DM patients under poor metabolic control significantly reduced fibrinogen concentrations in the α 2 α 2 and α 1 α 2 genotypes (P<0.001) of the Hind III polymorphism in the FGB 5' region (AKA -148C>T, in complete allelic association with -455G>A [32]) but not the α 1 α 1 genotype (P interaction <0.001), which they attributed to a relationship between the genotypes and glycaemic control (Fig 4D histogram). Fibrinogen levels were significantly higher before than after treatment (3.22 vs. 2.82 g/L), and an interpretation from the perspective of quantile-dependent expressivity attributes the results to the larger genetic effect before treatment (0.45 g/L per α 2 -allele) when fibrinogen concentrations were high vs. after treatment (0.05 g/L per α 2 -allele) when fibrinogen concentrations were lower [58].

Albuminuria
A strong correlation between urinary albumin excretion rate and fibrinogen plasma levels reported by Javorský et al. in T2DM patients (r = 0.48, p = 0.001) may reflect their common relationship with underlying inflammation [59]. The histogram in Fig 4E shows progressively increasing fibrinogen difference between macro and normoalbuminuria by the number of P-alleles of the lipoprotein lipase (LPL) PvuII polymorphism. However, the line graph shows that while average fibrinogen concentrations increased from 3.09 g/L in normoalbuminuria (urinary albumin excretion <20 mg/L), to 3.47 g/L in microalbuminuria (20-199 mg/L), to 4.37 g/L in macroalbuminuria (�200 mg/L), the fibrinogen difference between P-P-and P+P+ homozygotes was greater for macroalbuminia (1.11 g/L) than for microalbuminuria (0.53 g/L) or normoalbuminuria (0.75 g/L) [59], consistent with quantile-dependent expressivity.

Lipid-lowering drugs
Jang et al. [66] reported that fibrinogen concentrations were significantly higher in coronary heart disease patients who were not taking lipid-lowering drugs than those that were (>80% statins). Whereas the authors concluded that lipid-lowering drugs prevented the fibrinogenraising effect of the GG genotype of the IL-6 -572C>G polymorphisms (Fig 4F histogram), quantile-dependent expressivity suggests that the higher average fibrinogen concentrations of the untreated patients (line graph) could have contributed to the interaction.

Caveats and limitations
None of the SNPs identified to date explain any more than a few percent of fibrinogen heritability, which means that the effects of any particular SNP is not necessarily constrained by the results of Fig 1. Humphries et al. report [32] of a diminishing (not increasing) affect of the  [66] on differences between coronary heart disease patients on and off lipid lowering medications (Rx) by IL-6 -572C>G genotypes.
https://doi.org/10.1371/journal.pone.0262395.g004 FGB -455G>A polymorphism with age-related increases in men's average fibrinogen concentrations, and Rauramaa et al.'s [72] report of larger (not smaller) fibrinogen differences between FGA RsaI genotypes in the most physically active women with the lower average concentration vis-à-vis less-active women with higher average concentrations, are examples of environmental effect that are potentially inconsistent with quantile-dependent expressivity. We also acknowledge that the simple estimates of h 2 from Falconer formula [65] probably do not adequately describe fibrinogen inheritance, i.e., those derived from β OP may include shared environmental effects, and those derived from β FS may include shared environment and dominance effects and unmet restrictions on assortative mating. Finally, although there are multiple reports of interactions between the FGB -455G>A polymorphism and smoking, other reported gene-environment interaction generally lack replication.

Conclusion
Our analyses of the Framingham Heart Study cohorts suggest quantile-specific heritability of plasma fibrinogen concentrations. This was obtained by applying quantile regression to both offspring-parent and full-sib age-and sex-adjusted values. Heritability at the 90 th percentile of the fibrinogen distribution (0.65±0.08) was 2.2-fold greater than the heritability at the 10 th percentile (0.30±0.05). Quantile-dependent expressivity, and the selection of subjects from different portions of the fibrinogen distribution, potentially provides an alternative explanation for many gene-environment interactions reported for fibrinogen.